Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes (called ligands herein) include, for example, antigens, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding immunoassays, a labeled ligand, including immunocompetent derivatives and analogs of the ligand is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction proceeds as follows:
ligand+labeled ligand+receptor.fwdarw.
ligand-receptor+labeled ligand-receptor.
Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, coenzymes, inhibitors and allosteric effectors.
Consistent with the foregoing an immunoassay for carbamazepine in serum can be based on competition of an enzyme labeled carbamazepine analogue with carbamazepine in the patient serum for immobilized antibody binding sites.
Specific requirements for the labeled carbamazepine analogue include: 1) at least about 70-90% of the analogue can be bound by excess immobilized carbamazepine antibody; 2) affinity of the analogue for immobilized antibody is such that competition of a fixed amount of analogue with carbamazepine occurs in a therapeutically relevant concentration range; and 3) stability of the labeled carbamazepine analogue against hydrolysis of its enzyme label under storage conditions. Requirements imposed on the carbamazepine derivative include: 1) accessibility of the derivative to the immobilized antibody following conjugation with the enzyme label; 2) specific recognition of the analogue by the carbamazepine antibody; and 3) sufficient reactivity of the derivative with the enzyme label, either directly or following activation of the enzyme or derivative, under conditions that do not adversely affect enzyme activity.